Function Research Of AT-hook Gene (PgAHL1) In Panax Ginseng

Ginseng(Radix et Rhizoma Ginseng)is the root and rhizome of Panax ginseng CA Mey.,which has the effects of removing the spleen,benefiting the lung,and nourishing blood.It is our precious Chinese medicinal materials.The research of our group indicated that the"high quality"of P.ginseng(that is,active components represented by ginsenosides)can regulate it’s"excellent shape"(that is,the recognized traits of Daodi herbs).Rhizome is one of the important characteristics of P.ginseng.In order to further clarify the relationship between external properties and internal quality of P.ginseng,AHL(AT-hook nuclear localized protein,AHL)transcription factor that might participate in formation of rhizome was filtrated,and might be involved in regulation of ginsenosides biosynthesis at the same time,providing basis towards transcription factor participating in formation of"excellent shape and high quality"of P.ginseng.In this paper,50 PgAHLs were obtained,in which a PgAHL1 was filtrated to analyze its promotor structure and function.Results of transient expression in Nicotiana tabacum L.showed that PgAHL1F2,obtaining an element involved in the differentiation of palisade mesophyll cells,plays an important role in transcription regulation of PgAHL1.Meanwhile,UGT71A27(Pg＿S4157.4),a key enzyme gene of ginsenoside biosynthesis pathway was specifically expressed in rhizome,and its promotor had potential to bind with PgAHL1.This study lay foundation for further exploration towards PgAHLs regulates"excellent shape and high quality"of P.ginseng.The main research contents are as follows:1.PgAHLs gene expression and bioinformatics analysisAccording to bioinformatics analysis and screening,four genes homologous to Arabidopsis At AHL15were obtained in ginseng,named as PgAHL1,PgAHL2,PgAHL3 and PgAHL4respectively.The tissue expressional specificity of PgAHL1-PgAHL4 was analyzed according to a self-tested transcriptome data combined with real-time quantitative PCR.Results showed that PgAHL1 strongly expressed in rhizome,speculating that it may play an important role in regulation of rhizome development.2.Construction of PgAHL1 promoter expression vector A 1048 bp promoter region of PgAHL1 was cloned from lateral root DNA of P.ginseng.5′-terminal deletions fragment of the promoter was performed according to distribution of light-responsive regulatory element,circadian rhythm-responsive element,element participated in palisade mesophyll cell differentiation and transcription initiation site.Each fragment was recombined into a p CAMBIA1301:GUS plant expression vector,replacing original 35S promoter to construct a recombinant vector.3.PgAHL1 promoter region functional analysis Transgenic tobacco of PgAHL1pro and its deletion fragments were obtained by induction of Agrobacterium-mediated method,with expression activity detected by GUS staining Expression activity of GUS protein in the PgAHL1F2,containing an element involved in the differentiation of palisade mesophyll cells,had a significantly difference compared with CK~-.Therefore,it is speculated that the element involved in the differentiation of palisade mesophyll cells may play an important role in the regulational activity of PgAHL1.Regulation of PgAHL1 towards ginsenoside biosynthesis According to the expression profile combined with real-time quantitative PCR analysis,key enzyme genes PPDS(Pg＿S3293.6),UGT71A27(Pg＿S4157.4),CDP-MEK(Pg＿S2198.2,Pg＿S0550.8)of ginsenoside biosynthesis pathway were specifically expressed in rhizome of P.ginseng;cis-acting element analysis showed Pg＿S0550.8,Pg＿S2198.2,Pg＿S3293.6,Pg＿S4157.4containing AT-rich binding domains.Yeast one hybrid(Y1H)experiment indicated that AT-rich motif of Pg＿S4157.4 promoter can specifically bind with PgAHL1,preliminarily suggested that PgAHL1 affects biosynthesis of ginsenosides by regulating Pg＿S4157.4.